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Mix-and-Read, Bead- and Cell-Based Immunoassays Using the FMAT 8100 HTS System

t anti-mouse IgG are first coated with monoclonal anti- IL-8 antibodies. The coated beads and dye A-labeled anti-IL-8 detection antibodies are then added to the tissue culture media. An antibody/peptide sandwich will form and a fluorescent signal will be detected when the IL-8 peptide is present in the media and the bead/antibody/peptide complexes settle to the bottom of the well. To measure cell surface expression of ICAM-1, a dye B-labeled anti-ICAM-1 antibody is added simultaneously with the beads and IL-8 detection antibodies (Figures 1 and 2). Alternatively, HUVEC cells are detected by the addition of CentriRed Stain from Applied Biosystems (Figures 3 and 4). A standard curve, generated by addition of known amounts of IL-8 to wells containing untreated cells in tissue culture media, enables accurate determination of IL-8 concentration in test samples (Figure 5).

Only the fluorescence associated with the beads and cells is registered by the software. Because the depth of focus is within 100 μm from the well bottom, most of the unbound dyelabeled antibodies remain outside the focus area and are undetected, eliminating the need for wash steps.

To simultaneously quantitate IL-8 and cell surface ICAM-1 after TNF-α or IL-1 stimulation:

1.Plate 5,000 HUVEC cells/well in a 96-well TC-treated (tissue culture) plate.

2.Incubate overnight.

3.Replace media with 100 μL fresh media (with or without 10 ng/mL TNF-α or IL-1) and incubate overnight.

4.Add 50 μL PBS with .01% NaN3 containing:

Beads coated with capture monoclonal anti-IL-8 antibody

Dye A-labeled detection polyclonal anti-IL-8 antibody

Dye B-labeled anti-ICAM-1, antibody (Figures 1 and 2) or CentriRed Stain (Figures 3 and 4) 5.Incubate overnight at room temperature and scan.

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